Many cells contain a GTP-binding protein which is part of the adenylate cyclase complex. The GTP-binding protein is involved in the regulation of adenylate cyclase by hormones, cholera toxin, and other agents. We are attempting to isolate the nucleotide binding protein from porcine erythrocyte membranes. We are using a variant of S49 mouse lymphoma cells which are deficient in GTP-binding protein to assess the progress of the purification. Complementation of the S49 cell membranes deficient in GTP-binding protein with the factor was time- and temperature-dependent and enhanced by prior activation of the factor with choleragen and GTP or a nonhydrolyzable GTP analogue.